The objective of our research is to reveal the mechanisms underlying T cell-to-T cell transfer of HIV. This enigmatic means of viral spread may be central to our understanding HIV transmission and viral dissemination within the host. Recent studies indicate that adhesive contacts between infected and uninfected T cells, called virological synapses (VS), mediate a highly efficient mode of infection. VS are intercellular adhesive structures that are driven by Env engagement, cell signaling, actin rearrangements and recruitment of cell adhesion molecules. Despite anecdotal evidence supporting this mode of viral spread, studies have yet to rigorously examine how VS transmission fundamentally differs from cell-free infection. Given the high density of cells in the tissue sites, its role is likely to be central to the establishment and maintenance of HIV infection. A major impasse to the study of cell-to-cell transfer has been the absence of quantitative assays to assess the efficiency of cell-mediated infection. To study transmission of HIV at the VS, we have created a novel, fluorescent molecular clone of HIV, called HIV Gag-iGFP. Infection with the virus renders both the infected cells and the infectious particles highly fluorescent, allowing us to track viral assembly and transmission with extraordinary sensitivity. Using flow cytometry we estimate that VS-mediated viral transfer is 18,000-fold more efficient than uptake of cell-free virus. In contrast to cell-free exposure, VS-transferred virus is rapidly internalized into trypsin-resistant compartments. VS-mediated transfer requires Env-CD4 receptor interactions, but is not blocked by viral membrane fusion inhibitors or by patient-derived neutralizing antisera capable of blocking cell-free virus. This resistance to neutralization by patient antisera is dependent upon an intact cytoplasmic tail of Env. Quantitative live imaging of the VS reveals that HIV-expressing cells are polarized and make stable, Env-dependent contacts with target cells through uropod-like structures. With spinning disk confocal imaging we can track the recruitment of viral proteins to the synapse in producer cells and the movement of virus-containing vesicles while they bud into target T cells. In this proposal, we test the hypothesis that Env on the surface of infected cells is involved in cell signaling events that trigger T cell adhesion, activating viral assembly and transmission from cell to cell through a vesicular compartment. Understanding the cell biology of cell-cell spread will be essential to learning how to block these processes in vivo. We will therefore reveal how cell-surface Env triggers the coordinated assembly and transfer into HIV-naive T cells. The work has significance for chemotherapy, microbicide and vaccine development against HIV. PUBLIC HEALTH RELEVANCE: The HIV/AIDS pandemic affects over 40 million worldwide and over 1.2 million people in North America. HIV primarily replicates in CD4 helper T cells and can induce adhesive infection-promoting intercellular structures between these cells, which are called virological synapses (VS). A better understanding of VS-mediated viral spread, will allow us to devise novel strategies to inhibit HIV spread with new drugs, microbicides or vaccines.